Enterotoxigenic Escherichia coli (ETEC) is an important diarrheagenic pathogen in third world countries and is a biodefense threat since military personnel are likely to acquire this pathogen from non-sterile water and food sources in the field. ETEC is also responsible for a large number of fatalities in children. Vesicles derived from the outer membrane of Gram-negative bacteria have been detected in biopsies from human patients. Vesicle production is not unique to ETEC, in fact all gram-negative bacteria (including other biothreatening pathogens) produce vesicles, however they have been poorly characterized and little is known about the roles of vesicle components in disease. Whereas vesicles from nonpathogens are relatively harmless "empty shells", virulence proteins are associated with vesicles from pathogens such as ETEC. We have previously found that the majority of secreted physiologically active heat-labile enterotoxin (LT) is associated with ETEC vesicles. It is enriched in vesicles as compared to the cell and is present both inside and bound to the outside of the vesicle. Vesicles transfer virulence factors, such as LT, from the pathogen directly into host cells. LT on the surface of ETEC vesicles mediates vesicle binding, and binding causes subsequent internalization of vesicles by gut epithelial cells, leading to toxicity. The clinical consequence of enterotoxin entry into human intestinal cells is sudden water efflux or debilitating diarrhea. The overall objective of this research is to characterize the roles of vesicles and vesicle components in ETEC enterotoxin targeting and delivery into human intestinal cells. The aims of this proposal are to characterize the host cell interactions of purified ETEC vesicles, understand how the lipid and protein components of the vesicles affect cellular intoxication, and assess the impact of vesiculation levels in disease. We will utilize cell culture and confocal localization techniques, as well as an established in situ and in vivo models to sensitively and quantitatively characterize the roles of the toxic vesicle components. Previously identified mutations in E. coli genes that cause over- and underproduction of vesicles will be used in the models to evaluate how vesicle production correlates with toxicity. The data resulting from these aims will reveal key features of bacterial vesicles that function in human disease. These results will also broadly impact understanding the role of toxic vesicles produced by other pathogenic bacteria that threaten our defense capabilities.